Alkaloid glycoside for use as a medicament

ABSTRACT

The invention concerns an alkaloid glycoside for use in medicine. In a preferred aspect, the alkaloid glycoside is used for the stimulation of a class I-restricted immune response and/or a class II-restricted immune response. In a preferred aspect, the alkaloid glycoside is tomatine.

The present invention relates to a compound. More especially, thepresent invention relates to an adjuvant for use in a vaccine. In a moreparticular embodiment, the present invention relates to a glycosideadjuvant.

In the majority of vaccines used to date antibodies are raised againstproteins that are present on the surfaces of disease-causing(pathogenic) organisms (e.g. viruses, bacteria). Vaccine formulationstypically contain inactivated or killed pathogens. In many situations itis desirable to elicit an antibody response against a particular set ofproteins, peptides or carbohydrates that are present in the pathogen.This leads to a well-defined response which can be targeted againstproteins that are highly conserved between different strains of thepathogen, thereby achieving a broader-based immunity.

Furthermore, the ability to stimulate both class I- and classII-restricted immune responses is deemed to be important because itleads to a more comprehensive level of immunity than is currentlyachieved with existing vaccines.

Briefly, class II-restricted responses involve the production ofantibodies that are specific to the proteins or peptides used in thevaccine—this is often referred to as the humoral immune response.

Class I-restricted immune responses are cell-mediated responses that areresponsible for detecting and dealing with viral infections. In contrastto the class II-restricted responses, class I responses involve theactivation of CD8+ cytotoxic T lymphocytes (CTLs). These CTLs areactivated by oligomeric peptides that are derived from non-selfproteins. These peptides are presented at the surface of cells inconjunction with the products of the major histocompatibility complex I(MHC-1). Activated CTLs are able to recognise and destroy virus-infectedcells that have non-self oligopeptides on their surfaces.

The use of adjuvants in vaccine formulations leads to enhanced immuneresponses, particularly against soluble proteins and peptides which areonly weakly immunogenic.

Several adjuvants have been described in the literature (e.g. completeor incomplete Freund's adjuvant) but most are only of experimentalinterest because of their severe inflammatory side-effects. So far,saponin-based adjuvants are believed by some to be amongst the mostpromising because of their activity and their relatively low toxicityand inflammatory side-effects.

By way of example, EP-A-0231039 discloses the use of a saponin glycosideas an adjuvant for vaccines. These particular adjuvants, which are inthe form of nanoparticles, are characterised by their uniquearchitectures and are often referred to as immunostimulatory complexes(ISCOMS). These are produced by mixing a saponin glycoside andcholesterol in aqueous solutions, often in the presence of a lipid anddetergent, followed by one or more purification steps. The saponin ispresent in at least its critical micellar concentration (CMC). Theability of ISCOMS to elicit high antibody titres against a wide range ofantigenic proteins and peptides has been demonstrated as has theirability to induce CTL activity (i.e. the ISCOM nanoparticles canstimulate both class I- and class II-restricted immune responses).

However, there is still a continuing need to develop adjuvants forvaccines, in particular adjuvants that can stimulate class I-restrictedand/or class II-restricted immune responses to naturally occurring orsynthetic proteins and oligopeptides.

According to a first aspect of the present invention there is providedan alkaloid glycoside for use in medicine.

According to a second aspect of the present invention there is providedan alkaloid glycoside for the stimulation of a class I-restricted immuneresponse and/or a class II-restricted immune response.

According to a third aspect of the present invention there is providedan alkaloid glycoside for use as a vaccine adjuvant.

According to a fourth aspect of the present invention there is providedthe use of an alkaloid glycoside in the manufacture of a medicament foruse as a vaccine.

According to a fifth aspect of the present invention there is providedthe use of an alkaloid glycoside as an adjuvant in the manufacture of amedicament for use as a vaccine.

According to a sixth aspect of the present invention there is providedthe use of an alkaloid glycoside in the manufacture of a medicament tostimulate a class I-restricted immune response and/or a classII-restricted immune response.

According to a seventh aspect of the present invention there is provideda process of preparing a composition comprising admixing an agentcapable of inducing an immune response with an alkaloid glycoside,wherein the agent is in an amount to cause an induction of an immuneresponse and wherein the alkaloid glycoside is in an amount for use asan adjuvant.

According to an eighth aspect of the present invention there is provideda process of preparing a composition comprising admixing an agentcapable of inducing an immune response with an alkaloid glycoside,wherein the agent is in an amount to cause an induction of an immuneresponse and wherein the alkaloid glycoside is in an amount to causestimulation of a class I-restricted immune response and/or a classII-restricted immune response.

According to a ninth aspect of the present invention there is provided amethod of treatment, the method comprising administering to a subject acomposition wherein the composition comprises an agent capable ofinducing an immune response within the subject and wherein thecomposition also comprises an alkaloid glycoside, wherein the agent isin an amount to cause an induction of an immune response and wherein thealkaloid glycoside is in an amount for use as an adjuvant.

According to a tenth aspect of the present invention there is provided amethod of treatment, the method comprising administering to a subject analkaloid glycoside, wherein the alkaloid glycoside is in an amount tocause stimulation of a class I-restricted immune response and/or a classII-restricted immune response.

According to an eleventh aspect of the present invention there isprovided a method of treatment, the method comprising administering to asubject in need of treatment a composition wherein the compositioncomprises an agent capable of inducing an immune response within thesubject and wherein the composition also comprises an alkaloidglycoside, wherein the agent is in an amount to cause an induction animmune response and wherein the alkaloid glycoside is in an amount tocause stimulation of a class I-restricted immune response and/or a classII-restricted immune response.

According to a twelfth aspect of the present invention there is providedthe combination of an alkaloid glycoside and an agent capable ofinducing an immune response.

According to a thirteenth aspect of the present invention there isprovided the combination of two or more compositions for sequential orconsecutive administration to a subject, wherein at least onecomposition comprises an alkaloid glycoside and at least one othercomposition comprises an agent capable of inducing an immune response.

Thus, the present invention is based on the surprising finding that itis possible to use an alkaloid glycoside for a medical usuage, inparticular for the stimulation of a class I-restricted immune responseand/or a class II-restricted immune response. This finding is highlysurprising as R. Bomford, M. Stapleton, J. E. Beesley, E. A Jessup, K.R. Price, G. R. Fenwick (in “Adjuvanticity and ISCOM formation bystructurally divests saponins”; Vaccine, vol.10, (1992), 572-577.) statethat glycoalkaloids (i.e. alkaloid glycosides) lack adjuvant activity.

The term “subject” includes any one or more different types of animal,including humans. Preferably, the term means a human.

The term “glycoside” as used herein means a derivative of a sugar (whichmay be a simple sugar or a complex sugar) in which the hydroxyl groupattached to carbon 1 of the sugar is substituted by an alcoholic, orother, aglycone group.

In a preferred embodiment, the term “glycoside” as used herein means achemical entity/substance comprising a steroid, triterpene or otherpolycyclic aliphatic structure and one or more carbohydrates.

The term “alkaloid glycoside” as used herein means a glycoside capableof combining with acids to form salts.

In a preferred embodiment, the term “alkaloid glycoside” as used hereinmeans a glycoside where the aglycone group is a steroid, triterpene orother polycylic aliphatic structure which contains at least one nitrogenheterocycle (i.e. a heterocyclic group comprising at least N).

Preferably, the nitrogen heterocycle is a derivative of pyrrolidine,piperidine, imidazoline, piperazine, morpholine, pyridine, quinoline,isoquinoline, or pyrimidine.

Preferably the alkaloid glycoside is substantially (virtually) waterinsoluble.

Preferably, the term “substantially (virtually) water insoluble” meanshaving a solubility of less than 300 mg per 1000 cc H₂O.

Examples of suitable alkaloid glycosides may include tomatine, chaconinecommersonine, demissine, solanine and solasonine.

For the present invention the alkaloid glycoside may be a specificalkaloid glycoside or it may be a mixture of alkaloid glycosides.

Preferably, the alkaloid glycoside is at least tomatine (viz. (3β, 5α,22β, 25S)-Spirosolan-3-ylO-β-D-glucopyranosyl-(1-2)-O-[β-D-xlopyranosyl-(1-3)]-O-β-D-glucopyranosyl-(1-4)-β-D-galactopyranoside).

Preferably, the alkaloid glycoside is tomatine.

In each of the aspects of the present invention the alkaloid glycosidemay be used in combination with one or more other adjuvants, such assaponin glycoside.

In each of the aspects of the present invention the alkaloid glycosidemay be used in combination with one or more pharmaceutically acceptablecarrier(s) and/or diluent(s) and/or excipient(s).

In each of the aspects of the present invention the alkaloid glycosidemay be used in combination with one or more other active agent(s).

The composition of the present invention may be used for prophylactictreatment or for curative treatment.

Preferably, the composition of the present invention may be used forprophylactic treatment.

Preferably, the composition is a vaccine composition.

Preferably, the composition of the present invention is translucent.

Preferably the method of treatment is a method of vaccination orimmunisation.

In the method of treatment, the alkaloid glycoside may be administeredat—or delivered to—a different site on or in a subject than some or allof the agent that is capable of inducing an immune response.

Preferably, the immune response is at least partial vaccination orimmunisation.

More preferably, the immune response is substantial vaccination orimmunisation.

In a preferred aspect, the agent capable of inducing an immune responseis a protein or oligopeptide.

Typically, the protein or oligopeptide is isolatable from a virus, abacterium, a parasite, or an animal cell.

In a more preferred aspect, the agent capable of inducing an immuneresponse is a naturally occurring protein or oligopeptide or a syntheticprotein or oligopeptide.

Here the term “synthetic” includes proteins or oligopeptides made byrecombinant DNA techniques and/or proteins or oligopeptides made bysynthetic chemical techniques.

Preferably, the protein or oligopeptide is equivalent to a T cellepitope.

For some applications, the protein or oligopeptide may be water soluble.

Preferably, the term “water soluble” means ” means having a solubilityof at least 300 mg per 1000 cc H₂O.

For some applications, the protein or oligopeptide may be more watersoluble than the alkaloid glycoside.

More preferably the protein or oligopeptide is weakly immunogenic. Theterm “weakly immunogenic” means that when administered in the absence ofadjuvants, the agent induces less than a tenfold increase in antibodytitres or less than 45% cell lysis in an assay of CTL activity.

For some applications, preferably the protein or oligopeptide issubstantially purified prior to admixture with the alkaloid glycoside.

With the present invention, the alkaloid glycoside may be used incombination with one or more agents capable of inducing an immuneresponse.

For some applications, preferably the alkaloid glycoside is used incombination with one specific type of agent capable of inducing animmune response.

Preferably, the alkaloid glycoside of the present invention is used tostimulate both class I-restricted and class II-restricted immuneresponses.

More preferably, the alkaloid glycoside of the present invention is usedto stimulate both class I-restricted and class II-restricted immuneresponses to naturally occurring or synthetic proteins andoligopeptides.

In the combination aspect of the present invention, the alkaloidglycoside need not necessarily be admixed with the agent capable ofinducing an immune response. Preferably, however, the alkaloid glycosideis admixed with the agent capable of inducing an immune response.

In a preferred aspect, the alkaloid glycoside may be admixed with anagent that is capable of suppressing the haemolytic activity of thealkaloid glycoside.

A preferred agent that is capable of suppressing the haemolytic activityof the alkaloid glycoside is a sterol.

Preferred sterols that are capable of suppressing the haemolyticactivity of the alkaloid glycoside are sterols of animal or plant originand may include any one or more of cholesterol, stigmasterol,lumisterol.

A highly preferred agent that is capable of suppressing the haemolyticactivity of the alkaloid glycoside is cholesterol.

In a preferred aspect, the alkaloid glycoside may be admixed with asolubiliser for the alkaloid glycoside—particularly if the alkaloidglycoside is substantially insoluble in water.

In a preferred aspect, the solubiliser is a detergent.

Suitable detergents include any one or more of a non-ionic detergent, anionic detergent, and a zwitterionic detergent.

Preferably, the detergent is at least a non-ionic detergent.

Preferably, the detergent is a non-ionic detergent.

A preferred detergent is β-octylglucopyranoside.

If a detergent is used, then preferably the detergent is removed fromthe composition that is to be used for the method of treatment. In thisregard, the art is replete with techniques to remove detergents. By wayof example, the detergent is removed by dialysis, chromatography orgradient centrifugation. Preferably, the detergent is removed bydialysis.

The composition of the present invention may also include othercomponents—such as one or more lipids.

In a more preferred aspect, the composition of the present inventionthus comprises tomatine, cholesterol, a non-ionic detergent, and saline.Cholesterol is present to suppress the haemolytic activity of tomatine.Since tomatine is virtually insoluble in water, the non-ionic detergentis employed to solubilise it. The mixture is dialysed against saline toremove the detergent. In this way the adjuvant formulation is obtainedas a translucent dispersion.

With the present invention, the alkaloid glycoside may be administeredsubsequent to, and/or consecutive with, and/or prior to administrationof the agent capable of inducing an immune response. Preferably, atleast some of the alkaloid glycoside is administered consecutively withat least some of the agent capable of inducing an immune response.

For some applications, preferably at least a substantial proportion ofthe alkaloid glycoside is administered consecutively with at least asubstantial proportion of the agent capable of inducing an immuneresponse.

With the present invention the some or all of the alkaloid glycoside maybe administered in the same composition as some or all of the agentcapable of inducing an immune response.

Alternatively, the alkaloid glycoside may be administered in a differentcomposition as the agent capable of inducing an immune response.

Preferably, the alkaloid glycoside is administered in the samecomposition as the agent capable of inducing an immune response

The present invention will now be described by way of example only. Inthe following examples, room temperature was 20° C.

EXAMPLE 1

{Preparation of composition RAM-1*—which comprises an adjuvant accordingto the present invention and an agent capable of inducing an immuneresponse. }

Part A

25 mg tomatine, 3.1 mg phosphatidylethanolamine and 125 mgβ-octylglucopyranoside were added to 4 ml of saline (0.9% sodiumchloride solution). The mixture was warmed to 60° C. and shakenvigorously over a two hour period using a vortex mixture until a uniformfaintly cloudy dispersion was obtained. The mixture was allowed to coolto room temperature before use.

Part B

6.25 mg cholesterol, 3.1 mg phosphatidylethanolamine and 125.0 mgβ-octylglucopyranoside were added to 3 ml of saline (0.9% sodiumchloride solution). The mixture was warmed to 50° C. and shakenvigorously using a vortex mixer until a clear solution was obtained. Themixture was allowed to cool to room temperature before use.

Part C

25 mg ovalbumin (OVA) was dissolved in 3 ml of saline (0.9% sodiumchloride solution) at room temperature.

Part A was mixed with Part B and shaken vigorously over a period of twohours. To this mixture was added Part C followed by vigorous shaking.The mixture was placed in a thermostatted water bath at 35° C. for 12hours and shaken regularly during this period. After the mixture wasallowed to cool to room temperature it was placed in a dialysis tube (Mr10000 cut-off) and dialysed against saline for 24 hours to produce theadjuvant formulation RAM-1*.

EXAMPLE 2

{Preparation of composition RAM-1—which comprises an adjuvant accordingto the present invention (but no agent capable of inducing an immuneresponse). }

Part A

25 mg tomatine, 3.1 mg phosphatidylethanolamine and 125 mgβ-octylglucopyranoside were added to 4 ml of saline (0.9% sodiumchloride solution). The mixture was warmed to 60° C. and shakenvigorously over a 2 hour period using a vortex mixture until a uniformfaintly cloudy dispersion was obtained. The mixture was allowed to coolto room temperature before use.

Part B

6.25 mg cholesterol, 3.1 mg phosphatidylethanolamine and 125.0 mgβ-octylglucopyranoside were added to 3 ml of saline (0.9% sodiumchloride solution). The mixture was warmed to 50° C. and shakenvigorously using a vortex mixer until a clear solution was obtained. Themixture was allowed to cool to room temperature before use.

Part A was mixed with Part B and shaken vigorously over a period of twohours. To this mixture was added 3 ml of saline (0.9% sodium chloridesolution) followed by vigorous shaking. The mixture was placed in athermostatted water bath at 35° C. for 12 hours and shaken regularlyduring this period. After the mixture was allowed to cool to roomtemperature it was placed in a dialysis tube (Mr 10000 cut-off) anddialysed against saline for 24 hours to produce the adjuvant formulationRAM-1.

EXAMPLE 3

{Preparation of composition RAM-2*—which comprises an adjuvant accordingto the present invention and an agent capable of inducing an immuneresponse.}

Part A

99 mg tomatine, 12.5 mg phosphatidylethanolamine and 495 mgβ-octylglucopyranoside were added to 4 ml of saline (0.9% sodiumchloride solution). The mixture was warmed to 50° C. and shakenvigorously over a two hour period using a vortex mixer until a uniformfaintly cloudy dispersion was obtained. The mixture was allowed to coolto room temperature before use.

Part B

25 mg cholesterol, 12.5 mg phosphatidylethanolamine and 495 mgβ-octylglucopyranoside were added to 3 ml of saline (0.9% salinechloride solution). The mixture was warmed to 50° C. and shakenvigorously using a vortex mixer until a clear solution was obtained. Themixture was allowed to cool to room temperature before use.

Part C

10 mg ovalbumin (OVA) was dissolved in 3 ml of saline (0.9% sodiumchloride solution) at room temperature.

Part A was mixed with Part B and shaken vigorously over a period of twohours. To this mixture was added Part C followed by vigorous shaking.The mixture was placed in a thermostatted water bath at 35° C. for 12hours and shaken vigorously during this period. After the mixture wasallowed to cool to room temperature it was placed in a dialysis tube (Mr3000 cut-off) and dialysed against saline for 24 hours to produce theadjuvant formulation RAM-2*.

EXAMPLE 4

{Preparation of composition RAM-2—which comprises an adjuvant accordingto the present invention (but no agent capable of inducing an immuneresponse).}

Part A

99 mg tomatine, 12.5 mg phosphatidylethanolamine and 495 mgβ-octylglucopyranoside were added to 4 ml of saline (0.9% sodiumchloride solution). The mixture was warmed to 50° C. and shakenvigorously over a two hour period using a vortex mixer until a uniformfaintly cloudy dispersion was obtained. The mixture was allowed to coolto room temperature before use.

Part B

25 mg cholesterol, 12.5 mg phosphatidylsethanolamine and 494 mgβ-octylglucopyranoside were added to 3 ml of saline (0.9% sodiumchloride solution). The mixture was warmed to 50° C. and shakenvigorously using a vortex mixer until a clear solution was obtained. Themixture was added to cool to room temperature before use.

Part A was mixed with Part B and shaken vigorously over a period of twohours. To this mixture was added 3 ml of saline (0.9% sodium chloridesolution) followed by vigorous stirring. The mixture was placed in athermostatted water bath at 35° C. for 12 hours and shaken regularlyduring this period. After the mixture was allowed to cool to roomtemperature it was placed in a dialysis tube (Mr 3000 cut-off) anddialysed against saline for 24 hours to produce the adjuvant formulationRAM-2.

Antibody Titre Assays

Four sets of experiments were carried out as shown in Table 1. Eachgroup consisted of six mice.

TABLE 1 Group I Antigen given subcutaneously (s.c.) at t = 0 (noadjuvants) Group II RAM-1* or RAM-2* given s.c. at t = 0 Group III RAM-1or RAM-2 given s.c. at t = 0 followed by separate injection or antigenat different site 3 hours later Group IV RAM-1 or RAM-2 given s.c. at t= 0 (no antigen)

The immunisation/bleeding regime is shown Table 2 below.

TABLE 2 Immunisation 1st 2nd 3rd 4th — — Days 0 7 21 35 56 84 Weeks 0 13 5 8 12 Bleeding (pre) 1 2 3 4 5 6

The results of the experiments were as follows. Anti-OVA specificantibody titres in the blood serum obtained on day 35 from C57BL/6 miceare shown in Table 3. These titres were measured using standard ELISAprocedures and are expressed as logarithm to the base 10 of the highestdilution giving an optical density (OD) reading of >0.1. The backgroundOD was determined by taking the mean of samples from 6 non-immunizedmice.

TABLE 3 GROUP I GROUP II GROUP III GROUP IV log₁₀ antibody 2.7 6.0 3.32.0 titre

The results showed that all adjuvants lead to high titres ofantigen-specific antibodies during the course of the immunisation.Comparison of the data from the Group II and Group III experimentsindicate that the adjuvant effect can be achieved by independentadministration of antigen and antigen-free adjuvant formulation. None ofthe Group I or IV experiments showed increases in antigen-specificantibody titres.

Cytotoxicity Assays

Cytotoxic activity mediated by T lymphocytes was measured using theovalbumin-transfected EL4 (C57BL/6, H-2b thymoma) monoclonal murine cellline as a target. Untransfected EL4 cells were used as negativecontrols. Spleen cells (3×10⁷) from normal or immunised mice primed 14days earlier were incubated with 1.5×10⁶ irradiated (200 Gy) 15-12 cells(gp160 transfectants) or 3T3 cells (which served as controls) for 5days. Target cells (1×10⁶) were labelled with 100 μCi ⁵¹Cr. Target cellswere washed three times in RPMI 1640 medium, diluted to 2×10⁵ cells/ml,and 50μ (10⁴) of target cells added to each microtitre well containingantigen or peptide (10 μM) and preincubated for 30 min. Effector cellswere resuspended in complete mdium (CM) to yield a range of effector:target (E:T) ratios. Target cells (10⁴) were then incubated at differentE:T ratios in a total volume of 200 μl of 10% CM for an additional 4 h.Supernatants (100 μl) were collected and counted for ⁵¹Cr release.Spontaneous release and maximal release values were determined byincubating target cells in 10% CM alone or 0.1% Triton X-100. PercentCytotoxicity was calculated using the formula:

100×(ER−SR)/(MR−SR)

where ER is experimental release, SR is spontaneous release and ER ismaximal release. The results shown are shown in Table 4 and representthe means of triplicate determinations.

TABLE 4 % of Cytotoxic lysis E:T ratio antigen only alum + anitgenRAM-1* 100:1  45 12 60 50:1 30 4 40 25:1 19 3 33 12.5:1   — — 6

SUMMARY

In accordance with the present invention, we have found that alkaloidglycosides—such as tomatine—are able to elicit a strong class I- andclass II-restricted immune response when administered with solubleproteins.

Other modifications will be apparent to those skilled in the art. Forexample, solanine or chaconine may be used instead of or in addition totomatine. By way of further example, any one or more of: Gp120 fromhuman immunodeficiency virus, nucleoprotein from influenza virus, PIouter membrane protein from Neisseria gonorrhoeae, or 055:B5polysaccharide from Escherichia coli may be used instead of or inaddition to OVA.

What is claimed is:
 1. A medicine comprising one or more alkaloidglycosides and at least one agent which is a protein or oligopeptide,wherein one of the alkaloid glycosides is tomatine, and wherein theagent is capable of inducing an immune response.
 2. The medicine ofclaim 1 wherein the alkaloid glycoside is present in an amount for thestimulation of a class I-restricted immune response and/or a classII-restricted immune response.
 3. The medicine according to claim 1,wherein the agent capable of inducing an immune response is weaklyimmunogenic.
 4. The medicine of claim 1 wherein the medicine is avaccine or vaccine adjuvant.
 5. The medicine of claim 1, wherein themedicine stimulates a class I-restricted immune response and/or a classII-restricted immune response.